Therapeutic In Vivo Gene Editing Achieved by a Hypercompact CRISPR-Cas12f1 System Delivered with All-in-One Adeno-Associated Virus.

CRISPR-based gene therapies are making remarkable strides toward the clinic. But the large size of most widely used Cas endonucleases including Cas9 and Cas12a restricts their efficient delivery by the adeno-associated virus (AAV) for in vivo gene editing. Being exceptionally small, the recently engineered type V-F CRISPR-Cas12f1 systems can overcome the cargo packaging bottleneck and present as strong candidates for therapeutic applications. In this study, the pairwise editing efficiencies of different engineered Cas12f1/sgRNA scaffold combinations are systemically screened and optimized, and the CasMINI_v3.1/ge4.1 system is identified as being able to significantly boost the gene editing activity. Moreover, packaged into single AAV vectors and delivered via subretinal injection, CasMINI_v3.1/ge4.1 achieves remarkably high in vivo editing efficiencies, over 70% in transduced retinal cells. Further, the efficacy of this Cas12f1 system-based gene therapy to treat retinitis pigmentosa in RhoP23H mice is demonstrated by the therapeutic benefits achieved including rescued visual function and structural preservation. And minimal bystander editing activity is detected. This work advances and expands the therapeutic potential of the miniature Cas12f1 system to support efficient and accurate in vivo gene therapy.

Opening the Soul Window Manually: Limbal Tissue Scaffolds with Electrospun Polycaprolactone/Gelatin Nanocomposites.

Restricted by the difficulty in fabricating scaffolds suitable for cell proliferation, the use of ex vivo expanded limbal stem cell (LSC) for LSC transplantation, an effective treatment method for patients with limb stem cell deficiency (LSCD), is hard to be widely used in clinical practice. To tackle these challenges, a novel electrospun polycaprolactone (PCL)/gelatin nanocomposite is proposed to make 3D scaffolds for limbal niche cells (LNC) proliferation in vitro, which is a milestone in the treatment of diseases such as LSCD. PCL and gelatin in different weight ratios are dissolved in a mixed solvent, and then electrospinning and cross-linking are performed to prepare a scaffold for cell proliferation. The characterizations of the nanocomposites indicate that the gelatin content has a significant effect on its micro-morphology, thermal properties, crystallinity, degradation temperature, hydrophilicity, and mechanical properties. P8G2-C (PCL: gelatin = 80: 20, cross-linked), with smooth fibers and homogeneous pores, has better hydrophilicity, mechanical properties, and flexibility, so it can support LNC as cell proliferation assays revealed. This detailed investigation presented here demonstrates the feasibility of using PCL/gelatin nanocomposites electrospun fiber membranes as a limbus tissue engineering scaffold, which undoubtedly provide a new perspective for the development of tissue engineering field.