mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy.

Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g.

dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy.

Many disease-causing genes possess functionally equivalent counterparts, which are often expressed in distinct cell types. An attractive gene therapy approach for inherited disorders caused by mutations in such genes is to transcriptionally activate the appropriate counterpart(s) to compensate for the missing gene function. This approach offers key advantages over conventional gene therapies because it is mutation- and gene size-independent.

A gene therapy for inherited blindness using dCas9-VPR-mediated transcriptional activation.

Catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) enables activation of silent genes. Many disease genes have counterparts, which serve similar functions but are expressed in distinct cell types. One attractive option to compensate for the missing function of a defective gene could be to transcriptionally activate its functionally equivalent counterpart via dCas9-VPR.

Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering.

Precise genome engineering is instrumental for biomedical research and holds great promise for future therapeutic applications. Site-specific recombinases (SSRs) are valuable tools for genome engineering due to their exceptional ability to mediate precise excision, integration and inversion of genomic DNA in living systems. The ever-increasing complexity of genome manipulations and the desire to understand the DNA-binding specificity of these enzymes are driving efforts to identify novel SSR systems with unique properties.

TET3 is recruited by REST for context-specific hydroxymethylation and induction of gene expression.

Ten-eleven translocation hydroxylases (TET1-3) oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). In neurons, increased 5hmC levels within gene bodies correlate positively with gene expression. The mechanisms controlling TET activity and 5hmC levels are poorly understood.

Analysis of cell surface markers specific for transplantable rod photoreceptors.

Purpose: Transplantation of cells into retinas affected by degenerative diseases to replace dying photoreceptors represents a promising therapeutic approach. Young photoreceptors of 4-day-old mice show the highest capacity to integrate into the retinas of adult mice following grafting. Additional enrichment of these donor cells before transplantation with cell surface marker-dependent sorting methods further increases success rates.

Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration.

In vitro folding of G protein-coupled receptors into a detergent environment represents a promising strategy for obtaining sufficient amounts of functional receptor molecules for structural studies. Typically, these preparations exhibit a poor long-term stability especially at the required high protein concentration. Here, we report a protocol for the stabilization of the Escherichia coli-expressed and subsequently folded neuropeptide Y receptor type 2.