Precision and efficacy of RNA-guided DNA integration in high-expressing muscle loci.

Gene replacement therapies in genetic medicine primarily rely on adeno-associated viral (AAV) vectors for transgene expression. However, episomal expression can decline over time due to epigenetic silencing. CRISPR-based integration methods offer promise for long-term transgene insertion. While the development of transgene integration methods has made substantial progress, identifying optimal insertion loci remains challenging. Skeletal muscle is a promising tissue for gene replacement owing to the ease of access, relative proportion of body mass, the multinucleated nature of muscle, and the potential for reduced adverse effects. Leveraging endogenous promoters in skeletal muscle, we evaluated two high-expressing loci using homology-independent targeted integration (HITI) to integrate reporter or therapeutic genes in mouse myoblasts. We hijacked the muscle creatine kinase (Ckm) and myoglobin (Mb) promoters by co-delivering CRISPR-Cas9 and a donor plasmid with promoterless constructs encoding green fluorescent protein (GFP) or human Factor IX (hFIX). Additionally, we deeply profiled our genome and transcriptome outcomes from targeted integration and evaluated the safety of the proposed sites. This study introduces a proof-of-concept technology for achieving high-level therapeutic gene expression in skeletal muscle, with potential applications in targeted integration-based medicine and synthetic biology.

Early introduction of 3D modeling modules promotes the development of simulation skills in downstream biomedical engineering curricula.

BACKGROUND: Recent advancements in additive manufacturing have made 3D design a desirable skill in combating the historically slow development of biomedical products. Due to the broad applicability of additive manufacturing to biomedical engineering, 3D design and 3D printing are attractive educational tools for biomedical engineering students. However, due to the multidisciplinary nature of biomedical engineering, finding a suitable spot in the curriculum to teach students basic and application-based skills for 3D manufacturing is difficult. Furthermore, prior training in fundamental 3D design skills may be needed to support the use of application-based supplementary content. RESULTS: We designed a SolidWorks Simulations toolkit to complement a sophomore (2nd-year)-level Biomechanics course and distributed this assignment to students with and without prior training in 3D design delivered in an introductory biomedical engineering course. Using short videos, example-based problem solving, and step-by-step tutorials, students completed this as an extra-credit assignment and completed a survey gauging student opinion on SolidWorks and 3D design, confidence in each target skill, and the effectiveness of assignment delivery. The compilation of survey responses suggests that the assignment effectively increased positive responses in student opinion on interest in and likeliness to use SolidWorks in both groups. However, confidence in the target assignment skills was higher in the trained group and fewer problems occurred in operating SolidWorks for trained students. Further, analyzing the distribution of student grades with respect to survey responses suggests that responses had no relationship with initial class grade. CONCLUSION: These data collectively indicate that prior training provided to the students had a positive impact on the effectiveness of the assignment although increases in student opinion on the utility of 3D design were observed in both trained and untrained students. Our work has generated and identified a useful educational supplement to enrich existing biomedical engineering course materials with practical skills.

Characterization and Biocompatibility Assessment of Boron Nitride Magnesium Nanocomposites for Orthopedic Applications.

Magnesium (Mg) has been intensively studied as a promising alternative material to inert metallic alloys for orthopedic fixation devices due to its biodegradable nature inside the body and its favorable biocompatibility. However, the low mechanical strength and rapid corrosion of Mg in physiological environments represent the main challenges for the development of Mg-based devices for orthopedic applications. A possible solution to these limitations is the incorporation of a small content of biocompatible nanoparticles into the Mg matrix to increase strength and possibly corrosion resistance of the resulting nanocomposites. In this work, the effect of adding boron nitride (BN) nanoparticles (0.5 and 1.5 vol.%) on the mechanical properties, corrosion behavior, and biocompatibility of Mg-based nanocomposites was investigated. The properties of the nanocomposites fabricated using powder metallurgy methods were assessed using microstructure analyses, microhardness, compression tests, in vitro corrosion, contact angle, and cytotoxicity tests. A significant increase in the microhardness, strength, and corrosion rates of Mg-BN nanocomposites was detected compared with those of pure Mg (0% BN). Crystalline surface post-corrosion byproducts were detected and identified via SEM, EDX, and XRD. Biocompatibility assessments showed that the incorporation of BN nanoparticles had no significant impact on the cytotoxicity of Mg and samples were hydrophilic based on the contact angle results. These results confirm that the addition of BN nanoparticles to the Mg matrix can increase strength and corrosion resistance without influencing cytotoxicity in vitro. Further investigation into the chemical behavior of nanocomposites in physiological environments is needed to determine the potential impact of corrosive byproducts. Surface treatments and formulation methods that would increase the viability of these materials in vivo are also needed.

Delivery challenges for CRISPR-Cas9 genome editing for Duchenne muscular dystrophy.

Duchene muscular dystrophy (DMD) is an X-linked neuromuscular disorder that affects about one in every 5000 live male births. DMD is caused by mutations in the gene that codes for dystrophin, which is required for muscle membrane stabilization. The loss of functional dystrophin causes muscle degradation that leads to weakness, loss of ambulation, cardiac and respiratory complications, and eventually, premature death. Therapies to treat DMD have advanced in the past decade, with treatments in clinical trials and four exon-skipping drugs receiving conditional Food and Drug Administration approval. However, to date, no treatment has provided long-term correction. Gene editing has emerged as a promising approach to treating DMD. There is a wide range of tools, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and, most notably, RNA-guided enzymes from the bacterial adaptive immune system clustered regularly interspaced short palindromic repeats (CRISPR). Although challenges in using CRISPR for gene therapy in humans still abound, including safety and efficiency of delivery, the future for CRISPR gene editing for DMD is promising. This review will summarize the progress in CRISPR gene editing for DMD including key summaries of current approaches, delivery methodologies, and the challenges that gene editing still faces as well as prospective solutions.