DG9-conjugated morpholino rescues phenotype in SMA mice by reaching the CNS via a subcutaneous administration.

Antisense oligonucleotide-mediated (AO-mediated) therapy is a promising strategy to treat several neurological diseases, including spinal muscular atrophy (SMA). However, limited delivery to the CNS with AOs administered intravenously or subcutaneously is a major challenge. Here, we demonstrate a single subcutaneous administration of cell-penetrating peptide DG9 conjugated to an AO called phosphorodiamidate morpholino oligomer (PMO) reached the CNS and significantly prolonged the median survival compared with unconjugated PMO and R6G-PMO in a severe SMA mouse model. Treated mice exhibited substantially higher expression of full-length survival of motor neuron 2 in both the CNS and systemic tissues compared with nontreated and unmodified AO-treated mice. The treatment ameliorated the atrophic musculature and improved breathing function accompanied by improved muscle strength and innervation at the neuromuscular junction with no signs of apparent toxicity. We also demonstrated DG9-conjugated PMO localized in nuclei in the spinal cord and brain after subcutaneous injections. Our data identify DG9 peptide conjugation as a powerful way to improve the efficacy of AO-mediated splice modulation. Finally, DG9-PMO is a promising therapeutic option to treat SMA and other neurological diseases, overcoming the necessity for intrathecal injections and treating body-wide tissues without apparent toxicity.

In vitro maturation and in vivo stability of bioprinted human nasal cartilage.

The removal of skin cancer lesions on the nose often results in the loss of nasal cartilage. The cartilage loss is either surgically replaced with autologous cartilage or synthetic grafts. However, these replacement options come with donor-site morbidity and resorption issues. 3-dimensional (3D) bioprinting technology offers the opportunity to engineer anatomical-shaped autologous nasal cartilage grafts. The 3D bioprinted cartilage grafts need to embody a mechanically competent extracellular matrix (ECM) to allow for surgical suturing and resistance to contraction during scar tissue formation. We investigated the effect of culture period on ECM formation and mechanical properties of 3D bioprinted constructs of human nasal chondrocytes (hNC)-laden type I collagen hydrogel in vitro and in vivo. Tissue-engineered nasal cartilage constructs developed from hNC culture in clinically approved collagen type I and type III semi-permeable membrane scaffold served as control. The resulting 3D bioprinted engineered nasal cartilage constructs were comparable or better than the controls both in vitro and in vivo. This study demonstrates that 3D bioprinted constructs of engineered nasal cartilage are feasible options in nasal cartilage reconstructive surgeries.

CRISPR Therapeutics for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder with a prevalence of approximately 1 in 3500-5000 males. DMD manifests as childhood-onset muscle degeneration, followed by loss of ambulation, cardiomyopathy, and death in early adulthood due to a lack of functional dystrophin protein. Out-of-frame mutations in the dystrophin gene are the most common underlying cause of DMD. Gene editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising therapeutic for DMD, as it can permanently correct DMD mutations and thus restore the reading frame, allowing for the production of functional dystrophin. The specific mechanism of gene editing can vary based on a variety of factors such as the number of cuts generated by CRISPR, the presence of an exogenous DNA template, or the current cell cycle stage. CRISPR-mediated gene editing for DMD has been tested both in vitro and in vivo, with many of these studies discussed herein. Additionally, novel modifications to the CRISPR system such as base or prime editors allow for more precise gene editing. Despite recent advances, limitations remain including delivery efficiency, off-target mutagenesis, and long-term maintenance of dystrophin. Further studies focusing on safety and accuracy of the CRISPR system are necessary prior to clinical translation.

Time course of 3D fibrocartilage formation by expanded human meniscus fibrochondrocytes in hypoxia.

Adult human meniscus fibrocartilage is avascular and nonhealing after injury. Meniscus tissue engineering aims to replace injured meniscus with lab-grown fibrocartilage. Dynamic culture systems may be necessary to generate fibrocartilage of sufficient mechanical properties for implantation; however, the optimal static preculture conditions before initiation of dynamic culture are unknown. This study thus investigated the time course of fibrocartilage formation by human meniscus fibrochondrocytes on a three-dimensional biomaterial scaffold under various static conditions. Human meniscus fibrochondrocytes from partial meniscectomy were expanded to passage 1 (P1) or P2 (3.0 ± 0.4 and 6.5 ± 0.6 population doublings), seeded onto type I collagen scaffolds, and grown in hypoxia (HYP, 3% O2 ) or normoxia (NRX, 20% O2 ) for 3, 6, and 9 weeks. Mechanical properties were not different between P1 and P2 cell-based constructs. Mechanical properties were lower in HYP, increased continually in NRX only, and were positively correlated with glycosaminoglycan content and accumulation of hyaline cartilage-like matrix components. The most mechanically competent tissues (NRX/9 weeks) reached 1/5 of the native meniscus instantaneous compression modulus but had an increasingly hypertrophic matrix-forming phenotype. HYP consistently suppressed the hypertrophic phenotype. The results provide baselines of engineered meniscus fibrocartilage properties under static conditions, which can be used to select a preculture strategy for dynamic culture depending on the desired combination of mechanical properties, hyaline cartilage-like matrix abundance, and hypertrophic phenotype.

eSkip-Finder: a machine learning-based web application and database to identify the optimal sequences of antisense oligonucleotides for exon skipping.

Exon skipping using antisense oligonucleotides (ASOs) has recently proven to be a powerful tool for mRNA splicing modulation. Several exon-skipping ASOs have been approved to treat genetic diseases worldwide. However, a significant challenge is the difficulty in selecting an optimal sequence for exon skipping. The efficacy of ASOs is often unpredictable, because of the numerous factors involved in exon skipping. To address this gap, we have developed a computational method using machine-learning algorithms that factors in many parameters as well as experimental data to design highly effective ASOs for exon skipping. eSkip-Finder (https://eskip-finder.org) is the first web-based resource for helping researchers identify effective exon skipping ASOs. eSkip-Finder features two sections: (i) a predictor of the exon skipping efficacy of novel ASOs and (ii) a database of exon skipping ASOs. The predictor facilitates rapid analysis of a given set of exon/intron sequences and ASO lengths to identify effective ASOs for exon skipping based on a machine learning model trained by experimental data. We confirmed that predictions correlated well with in vitro skipping efficacy of sequences that were not included in the training data. The database enables users to search for ASOs using queries such as gene name, species, and exon number.

Bioprinting of human nasoseptal chondrocytes-laden collagen hydrogel for cartilage tissue engineering.

Skin cancer patients often have tumorigenic lesions on their noses. Surgical resection of the lesions often results in nasal cartilage removal. Cartilage grafts taken from other anatomical sites are used for the surgical reconstruction of the nasal cartilage, but donor-site morbidity is a common problem. Autologous tissue-engineered nasal cartilage grafts can mitigate the problem, but commercially available scaffolds define the shape and sizes of the engineered grafts during tissue fabrication. Moreover, the engineered grafts suffer from the inhomogeneous distribution of the functional matrix of cartilage. Advances in 3D bioprinting technology offer the opportunity to engineer cartilages with customizable dimensions and anatomically shaped configurations without the inhomogeneous distribution of cartilage matrix. Here, we report the fidelity of Freeform Reversible Embedding of Suspended Hydrogel (FRESH) bioprinting as a strategy to generate customizable and homogenously distributed functional cartilage matrix engineered nasal cartilage. Using FRESH and in vitro chondrogenesis, we have fabricated tissue-engineered nasal cartilage from combining bovine type I collagen hydrogel and human nasoseptal chondrocytes. The engineered nasal cartilage constructs displayed molecular, biochemical and histological characteristics akin to native human nasal cartilage.

Restoration of dystrophin expression and correction of Duchenne muscular dystrophy by genome editing.

Introduction: Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder that affects approximately one in 3500-5000 male births. Patients experience muscle degeneration, loss of ambulation, and eventual death from cardiac or respiratory failure in early adulthood due to a lack of functional dystrophin protein, which is required to maintain the integrity of muscle cell membranes. Out-of-frame mutations in the DMD gene generally lead to no dystrophin protein expression and a more severe phenotype (DMD). Conversely, in-frame mutations are often associated with milder Becker muscular dystrophy (BMD) with a truncated dystrophin expression.Areas covered: Genome editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system can induce permanent corrections of the DMD gene, thus becoming an increasingly popular potential therapeutic method. In this review, we outline recent developments in CRISPR/Cas9 genome editing for the correction of DMD, both in vitro and in vivo, as well as novel delivery methods.Expert opinion: Despite recent advances, many limitations to CRISPR/Cas9 therapy are still prevalent such as off-target editing and immunogenicity. Specifically, for DMD, intervention time and efficient delivery to cardiac and skeletal muscles also present inherent challenges. Research needs to focus on the therapeutic safety and efficacy of this approach.