In vivo peptide-based delivery of a gene-modifying enzyme into cells of the central nervous system

Abstract

We report on the successful delivery of the Cre recombinase enzyme in the neural cells of mice in vivo by simple coinjection with peptides derived from HIV-TAT. Cre delivery activates the expression of a reporter gene in both neurons and astrocytes of the cortex without tissue damage and with a transduction efficiency that parallels or exceeds that of a commonly used adeno-associated virus. Our data indicate that the delivery peptides mediate efficient endosomal leakage and cytosolic escape in cells that have endocytosed Cre. The peptides, therefore, act in trans and do not require conjugation to the payload, greatly simplifying sample preparation. Moreover, the delivery peptides are exclusively composed of natural amino acids and are consequently readily degradable and processed by cells. We envision that this approach will be beneficial to applications that require the transient introduction of proteins into cells in vivo.

Elucidating the Impact of Payload Conjugation on the Cell-Penetrating Efficiency of the Endosomal Escape Peptide dfTAT: Implications for Future Designs for CPP-Based Delivery Systems

“…Recently, to facilitate quantification, we have used AlexaFluor 488-labeled Histone H1 (AF488-H1) as a probe of endosomal escape. , When co-incubated with dfTAT-like CPPs, AF488-H1 enters cells with minimal degradation and intrinsically accumulates in nuclei. The number of cells that display nuclear staining can then be counted (above a detection threshold) and a ratio of cytoplasmic/endosomal versus nuclear signal established . Molecules 1 , 2 , 3 , 4 , or 5 (5 μM) were co-incubated with AF488-H1 (1.25 μM) and administered to MDA-MB-231 cells (Figure A).…”

“…The fluorescence that remains trapped in the endosomes is also partially masked. Recently, to facilitate quantification, we have used AlexaFluor 488-labeled Histone H1 (AF488-H1) as a probe of endosomal escape. , When co-incubated with dfTAT-like CPPs, AF488-H1 enters cells with minimal degradation and intrinsically accumulates in nuclei. The number of cells that display nuclear staining can then be counted (above a detection threshold) and a ratio of cytoplasmic/endosomal versus nuclear signal established .…”

“…An example reproduced in many laboratories is that of TAT-Cre recombinase . In our experience, delivery of TAT-Cre into cells requires exposure times of 16 to 24h, as opposed to the 1h used herein with dfTAT . It is therefore possible that multiple mechanisms of endosomal escape exist: some slow, some fast, and some compatible with large payloads.…”

“…The concentration of the payload can also be titrated independently of that of dfTAT, leading to relative control of how many molecules of the payload enter the cell. We have recently extended this approach to the in vivo setting, using stereotactic cortical injections of mouse brains . The co-incubation format was exploited for the successful delivery of Cre recombinase into neurons and astrocytes.…”

Biogenic materials for CRISPR delivery and therapeutics

“…109 Furthermore, according to another study, Cre recombinase enzyme can be successfully delivered into mouse neural cells by simple co-injection of HIV-TAT-derived peptides, and its transduction efficiency is comparable to that of AAV. 110 Peptides and their hybrids can also be chemically designed to achieve effective gene editing. PPABLG is a cationic polypeptide that could perform the dual function of an efficient gene carrier and a cell-membrane-penetrating agent.…”