Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes.

Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.

In vivo human T cell engineering with enveloped delivery vehicles.

Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here, we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR-Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody-antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility.

Consistent Measurement of Parasite-Specific Antigen Levels in Sera of Patients with Neurocysticercosis Using Two Different Monoclonal Antibody (mAb)-Based Enzyme-Linked Immunosorbent Assays.

Monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) is a complementary diagnosis technique for neurocysticercosis (NCC), which detects circulating parasite antigen (Ag) indicative of viable infection and Ag levels that correlate well with the parasite burden. In this study, we compared the performance of two Ag-ELISA techniques for the detection of NCC. We assessed the agreement between our in-house TsW8/TsW5 Ag-ELISA and the widely used B158/B60 Ag-ELISA for measuring T. solium antigen levels in the sera from 113 patients with calcified, parenchymal, and subarachnoid NCC. Concordance was demonstrated evaluating the limits of agreement (LoAs) stratified by the type of NCC. Both ELISA's detected 47/48 (97.8%) subarachnoid NCC cases. In parenchymal and calcified NCC, the B158/B60 Ag-ELISA detected 19/24 (79.2%) and 18/41 (43.9%) cases, while the TsW8/TsW5 Ag-ELISA detected 21/24 (87.5%) and 13/41 (31.7%), respectively. Parenchymal and calcified NCC obtained a perfect agreement (100%), indicating that all sample results were within the predicted LoA, while for subarachnoid NCC, the agreement was 89.6%. The high concordance between the assays was confirmed by Lin's concordance coefficient (LCC = 0.97). Patients with viable parenchymal NCC (LCC = 0.95) obtained the highest concordance between assays, followed by subarachnoid NCC (LCC = 0.93) and calcified NCC (LCC = 0.92). The TsW8/TsW5 Ag-ELISA and B158/B60 Ag-ELISA showed high Ag measurement correlations across diverse types of NCC.

CRISPR-Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells.

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei.

Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering.

As genome engineering advances cell-based therapies, a versatile approach to introducing both CRISPR-Cas9 ribonucleoproteins (RNPs) and therapeutic transgenes into specific cells would be transformative. Autologous T cells expressing a chimeric antigen receptor (CAR) manufactured by viral transduction are approved to treat multiple blood cancers, but additional genetic modifications to alter cell programs will likely be required to treat solid tumors and for allogeneic cellular therapies. We have developed a one-step strategy using engineered lentiviral particles to introduce Cas9 RNPs and a CAR transgene into primary human T cells without electroporation. Furthermore, programming particle tropism allows us to target a specific cell type within a mixed cell population. As a proof-of-concept, we show that HIV-1 envelope targeted particles to edit CD4+ cells while sparing co-cultured CD8+ cells. This adaptable approach to immune cell engineering ex vivo provides a strategy applicable to the genetic modification of targeted somatic cells in vivo.

Engineering of monosized lipid-coated mesoporous silica nanoparticles for CRISPR delivery.

CRISPR gene editing technology is strategically foreseen to control diseases by correcting underlying aberrant genetic sequences. In order to overcome drawbacks associated with viral vectors, the establishment of an effective non-viral CRISPR delivery vehicle has become an important goal for nanomaterial scientists. Herein, we introduce a monosized lipid-coated mesoporous silica nanoparticle (LC-MSN) delivery vehicle that enables both loading of CRISPR components [145 µg ribonucleoprotein (RNP) or 40 µg plasmid/mg nanoparticles] and efficient release within cancer cells (70%). The RNP-loaded LC-MSN exhibited 10% gene editing in both in vitro reporter cancer cell lines and in an in vivo Ai9-tdTomato reporter mouse model. The structural and chemical versatility of the mesoporous silica core and lipid coating along with framework dissolution-assisted cargo delivery open new prospects towards safe CRISPR component delivery and enhanced gene editing. STATEMENT OF SIGNIFICANCE: After the discovery of CRISPR gene-correcting technology in bacteria. The translation of this technology to mammalian cells may change the face of cancer therapy within the next years. This was first made possible through the use of viral vectors; however, such systems limit the safe translation of CRISPR into clinics because its difficult preparation and immunogenicity. Therefore, biocompatible non-viral nanoparticulate systems are required to successfully deliver CRISPR into cancer cells. The present study presents the use of biomimetic lipid-coated mesoporous silica nanoparticles showing successful delivery of CRISPR ribonucleoprotein and plasmid into HeLa cervical and A549 lung cancer cells as well as successful gene editing in mice brain.

Use of magnetic particles in the purification of IgM antibodies against Taenia solium. / Uso de partículas magnéticas en la purificación de anticuerpos IgM contra Taenia solium.

The use of L protein coupled magnetic particles for the concentration and purification of immunoglobulin M (mIgM) monoclonal antibodies against Taenia solium was evaluated. Three concentration methods and different elution times were evaluated and the ratio of particles to the ratio of mIgM was optimized. It is demonstrated that: 1) with the use of magnetic particles, a previous concentration of mIgM is not required, which reduces the manipulation of the antibodies and improves the recovery, 2) the use of a binding buffer can be omitted, since the pH of most cell culture supernatants are neutral, and 3) longer elution times (~ 45 minutes) are needed to increase recovery to a level greater than 80%. The study demonstrates that the use of L protein-coupled magnetic particles is a simple and efficient tool for mIgM concentration and purification.

Se evaluó el uso de partículas magnéticas acopladas a proteína L para la concentración y purificación de anticuerpos monoclonales inmunoglobulina M (mIgM) contra Taenia solium. Se evaluaron tres métodos de concentración y diferentes tiempos de elución y se optimizó la proporción de partículas a la proporción de mIgM. Demostramos que: 1) con el uso partículas magnéticas no se requiere de una concentración previa de mIgM, lo que disminuye la manipulación de los anticuerpos y mejora la recuperación, 2) se puede omitir el uso de un tampón de unión, ya que el pH de la mayoría de los sobrenadantes de cultivo celular son neutros, y 3) se necesitan tiempos de elución más largos (~45 minutos) para aumentar la recuperación a un nivel mayor a 80%. El estudio demuestra que el uso de partículas magnéticas acopladas a proteína L es una herramienta simple y eficiente para la concentración y purificación de mIgM.

Uso de partículas magnéticas en la purificación de anticuerpos IgM contra Taenia solium / Use of magnetic particles in the purification of IgM antibodies against Taenia solium

RESUMEN Se evaluó el uso de partículas magnéticas acopladas a proteína L para la concentración y purificación de anticuerpos monoclonales inmunoglobulina M (mIgM) contra Taenia solium. Se evaluaron tres métodos de concentración y diferentes tiempos de elución y se optimizó la proporción de partículas a la proporción de mIgM. Demostramos que: 1) con el uso partículas magnéticas no se requiere de una concentración previa de mIgM, lo que disminuye la manipulación de los anticuerpos y mejora la recuperación, 2) se puede omitir el uso de un tampón de unión, ya que el pH de la mayoría de los sobrenadantes de cultivo celular son neutros, y 3) se necesitan tiempos de elución más largos (~45 minutos) para aumentar la recuperación a un nivel mayor a 80%. El estudio demuestra que el uso de partículas magnéticas acopladas a proteína L es una herramienta simple y eficiente para la concentración y purificación de mIgM.

ABSTRACT The use of L protein coupled magnetic particles for the concentration and purification of immunoglobulin M (mIgM) monoclonal antibodies against Taenia solium was evaluated. Three concentration methods and different elution times were evaluated and the ratio of particles to the ratio of mIgM was optimized. It is demonstrated that: 1) with the use of magnetic particles, a previous concentration of mIgM is not required, which reduces the manipulation of the antibodies and improves the recovery, 2) the use of a binding buffer can be omitted, since the pH of most cell culture supernatants are neutral, and 3) longer elution times (~ 45 minutes) are needed to increase recovery to a level greater than 80%. The study demonstrates that the use of L protein-coupled magnetic particles is a simple and efficient tool for mIgM concentration and purification.

Actinomicetos con actividad anti-candida aisladas de hormigas cortadoras de hojas Atta cephalotes (Formicidae: Myrmicinae: Attini) / Actinomyces with anti-candida activity isolated from leaf-cutting ants Atta cephalotes (Formicidae: Myrmicinae: Attini)

RESUMEN Objetivos. Aislar, seleccionar e identificar actinomicetos asociados a hormigas cortadoras de hojas Atta cephalotes (Linnaeus, 1758), que presenten mayor actividad anti-Candida. Materiales y métodos. Estudio transversal realizado en hormigas recolectadas de una localidad de Huánuco, Perú, a partir de las cuales se aislaron cepas de actinomicetos que fueron evaluadas mediante pruebas in vitro para determinar su capacidad antagonista frente a especies de Candida. Los actinomicetos de mayor antagonismo fueron seleccionados y cultivados en agitación, luego se obtuvieron los metabolitos extracelulares con solventes orgánicos y finalmente se evaluaron los extractos crudos para determinar cuantitativamente la concentración mínima inhibitoria (CMI). Resultados. Se logró aislar 30 actinomicetos, de los cuales el 47 % presentaron actividad antagonista a Candida albicans (C. albicans) ATCC 7516, el 43 % a Candida parapsilosis ATCC 7307, el 37% a Candida tropicalis ATCC 7206 y C. albicans ATCC 10231 y el 30% a C. albicans ATCC 98028. Extractos orgánicos de las cepas HAA-16 y HAA-17 presentaron marcada actividad anti-Candida; siendo el extracto de acetato de etilo de la cepa HAA17 el de mejor rendimiento por tener mayor espectro de actividad y presentar una CMI de 3,25 mg/ml frente a C. albicans ATCC 7516 y Candida parapsilosis ATCC 7307. Los actinomicetos seleccionados se identificaron mediante técnicas moleculares como miembros del género Streptomyces. Conclusiones. Los actinomicetos asociados a Atta cephalotes son excelentes productores de compuestos bioactivos, capaces de inhibir el crecimiento de levaduras patógenas del género Candida y con potencial aplicación en el desarrollo de nuevos productos naturales de interés biomédico.

ABSTRACT Objectives. To isolate, select and identify actinomyces associated to leaf-cutting ants Atta cephalotes (Linnaeus, 1758), that present a greater anti-Candida activity. Materials and Methods. Cross-sectional study made with ants collected at a location in Huánuco, Peru, from which strands of actinomyces were isolated and later evaluated by in vitro testing in order to determine its antagonistic capacity against species of Candida. The actinomyces with greater antagonism were selected and cultured by agitation, then the reliable extracellular metabolites were obtained with organic solvents, and finally the crude extracts were evaluated to determine quantitatively the minimum inhibiting concentration (MIC). Results. Thirty (30) actinomyces were isolated, of which 47% exhibited antagonistic activity against Candida albicans (C. albicans) ATCC 7516, 43% to Candida parapsilosis ATCC 7307, 37% to Candida tropicalis ATCC 7206 and C. albicans ATCC 10231, and 30% to C. albicans ATCC 98028. Organic extracts of the HAA-16 and HAA-17 strands exhibited noticeable anti-Candida activity, being the ethyl acetate extract of the HAA-17 strand the one with the highest performance thanks to a wider activity spectrum of MIC 3.25 mg/mL against C. albicans ATCC 7516 and Candida parapsilosis ATCC 7307. The selected actinomyces were identified by means of molecular techniques as members of the Streptomyces genus. Conclusions. Actinomyces associated to Atta cephalotes are excellent producers of bioactive compounds, being able to inhibit the growth of pathogenic mold of the Candida genus and with potential for application in the development of new natural products for the biomedical field.

[Actinomyces with anti-candida activity isolated from leaf-cutting ants Atta cephalotes (Formicidae: Myrmicinae: Attini)]. / Actinomicetos con actividad anti-candida aisladas de hormigas cortadoras de hojas Atta cephalotes (Formicidae: Myrmicinae: Attini).

OBJECTIVES.: To isolate, select and identify actinomyces associated to leaf-cutting ants Atta cephalotes (Linnaeus, 1758), that present a greater anti-Candida activity. MATERIALS AND METHODS.: Cross-sectional study made with ants collected at a location in Huánuco, Peru, from which strands of actinomyces were isolated and later evaluated by in vitro testing in order to determine its antagonistic capacity against species of Candida. The actinomyces with greater antagonism were selected and cultured by agitation, then the reliable extracellular metabolites were obtained with organic solvents, and finally the crude extracts were evaluated to determine quantitatively the minimum inhibiting concentration (MIC). RESULTS.: Thirty (30) actinomyces were isolated, of which 47% exhibited antagonistic activity against Candida albicans (C. albicans) ATCC 7516, 43% to Candida parapsilosis ATCC 7307, 37% to Candida tropicalis ATCC 7206 and C. albicans ATCC 10231, and 30% to C. albicans ATCC 98028. Organic extracts of the HAA-16 and HAA-17 strands exhibited noticeable anti-Candida activity, being the ethyl acetate extract of the HAA-17 strand the one with the highest performance thanks to a wider activity spectrum of MIC 3.25 mg/mL against C. albicans ATCC 7516 and Candida parapsilosis ATCC 7307. The selected actinomyces were identified by means of molecular techniques as members of the Streptomyces genus. CONCLUSIONS.: Actinomyces associated to Atta cephalotes are excellent producers of bioactive compounds, being able to inhibit the growth of pathogenic mold of the Candida genus and with potential for application in the development of new natural products for the biomedical field.

OBJETIVOS.: Aislar, seleccionar e identificar actinomicetos asociados a hormigas cortadoras de hojas Atta cephalotes (Linnaeus, 1758), que presenten mayor actividad anti-Candida. MATERIALES Y MÉTODOS.: Estudio transversal realizado en hormigas recolectadas de una localidad de Huánuco, Perú, a partir de las cuales se aislaron cepas de actinomicetos que fueron evaluadas mediante pruebas in vitro para determinar su capacidad antagonista frente a especies de Candida. Los actinomicetos de mayor antagonismo fueron seleccionados y cultivados en agitación, luego se obtuvieron los metabolitos extracelulares con solventes orgánicos y finalmente se evaluaron los extractos crudos para determinar cuantitativamente la concentración mínima inhibitoria (CMI). RESULTADOS.: Se logró aislar 30 actinomicetos, de los cuales el 47 % presentaron actividad antagonista a Candida albicans (C. albicans) ATCC 7516, el 43 % a Candida parapsilosis ATCC 7307, el 37% a Candida tropicalis ATCC 7206 y C. albicans ATCC 10231 y el 30% a C. albicans ATCC 98028. Extractos orgánicos de las cepas HAA-16 y HAA-17 presentaron marcada actividad anti-Candida; siendo el extracto de acetato de etilo de la cepa HAA17 el de mejor rendimiento por tener mayor espectro de actividad y presentar una CMI de 3,25 mg/ml frente a C. albicans ATCC 7516 y Candida parapsilosis ATCC 7307. Los actinomicetos seleccionados se identificaron mediante técnicas moleculares como miembros del género Streptomyces. CONCLUSIONES.: Los actinomicetos asociados a Atta cephalotes son excelentes productores de compuestos bioactivos, capaces de inhibir el crecimiento de levaduras patógenas del género Candida y con potencial aplicación en el desarrollo de nuevos productos naturales de interés biomédico.