CRISPR-Mediated Induction of Neuron-Enriched Mitochondrial Proteins Boosts Direct Glia-to-Neuron Conversion.

Astrocyte-to-neuron conversion is a promising avenue for neuronal replacement therapy. Neurons are particularly dependent on mitochondrial function, but how well mitochondria adapt to the new fate is unknown. Here, we determined the comprehensive mitochondrial proteome of cortical astrocytes and neurons, identifying about 150 significantly enriched mitochondrial proteins for each cell type, including transporters, metabolic enzymes, and cell-type-specific antioxidants. Monitoring their transition during reprogramming revealed late and only partial adaptation to the neuronal identity. Early dCas9-mediated activation of genes encoding mitochondrial proteins significantly improved conversion efficiency, particularly for neuron-enriched but not astrocyte-enriched antioxidant proteins. For example, Sod1 not only improves the survival of the converted neurons but also elicits a faster conversion pace, indicating that mitochondrial proteins act as enablers and drivers in this process. Transcriptional engineering of mitochondrial proteins with other functions improved reprogramming as well, demonstrating a broader role of mitochondrial proteins during fate conversion.

Deciphering the Role of Chondroitin Sulfate in Increasing the Transfection Efficiency of Amphipathic Peptide-Based Nanocomplexes.

Glycosaminoglycans, both cell-surface and exogenous, can interfere with DNA delivery efficiency of nonviral carrier systems. In this work, we report an extensive comparative study to explore the effect of exogenously added chondroitin sulfate on biophysical characteristics, cellular uptake, transfection efficiency, and intracellular trafficking of nanocomplexes formed using primary and secondary amphipathic peptides developed in our laboratory. Our results indicate that the presence of exogenous chondroitin sulfate exhibits differential enhancement in transfection efficiency of the amphipathic peptides depending upon their chemical nature. The enhancement was more pronounced in primary amphipathic peptide-based nanocomplexes as compared to the secondary counterpart. This difference can be attributed to possible alteration of the intracellular entry pathway in addition to increased extracellular stability, less cellular toxicity, and assistance in nuclear accumulation. These results imply potential use of glycosaminoglycans such as chondroitin sulfate to improve the transfection efficiency of primary amphipathic peptides for possible in vivo applications.

Efficient Cellular Entry of (r-x-r)-Type Carbamate-Plasmid DNA Complexes and Its Implication for Noninvasive Topical DNA Delivery to Skin.

Arginine-rich cell penetrating peptides are powerful tools for in vitro as well as in vivo delivery of a wide plethora of biomolecules. However, presence of consecutive arginine residues leads to enhanced amenability for proteolytic degradation as well as steric hindrances for membrane interactions which compromise its bioavailability. In order to overcome these limitations we previously reported a safe and stable octaarginine based oligomer, i.e., (r-x-r)4-carbamate, where the backbone amide linkages were replaced by carbamate linkages and 6-aminohexanoic acid based spacer moieties were incorporated for better flexibility, hydrophobicity, optimal spacing of guanidinium groups, and protection against proteolytic cleavage; resulting in improved transfection efficiency over its amide counterpart. In the present work we have investigated the mechanism behind this enhanced transfection efficiency and, based on our observations, demonstrate how the synergistic effect of rationalized oligomer designing, complex characteristics, and cell type contributes to overall effective intracellular delivery. Our results indicate that the (r-x-r)4-carbamate-plasmid DNA complexes primarily utilize lipid raft dependent pathway of cellular entry more than other pathways, and this possibly facilitates their increased entry in the lipid raft rich milieu of skin cells. We also emphasize the utility of oligomer (r-x-r)4-carbamate as an efficient carrier for topical delivery of nucleic acids in skin tissue. This carrier can be utilized for safe, efficient, and noninvasive delivery of therapeutically relevant macromolecular hydrophilic cargo like nucleic acids to skin.

Non-invasive topical delivery of plasmid DNA to the skin using a peptide carrier.

Topical delivery to skin is an essential step in non-invasive application of nucleic acid therapeutics for cutaneous disorders. The barrier posed by different layers of the skin - stratum corneum on top followed by the viable epidermis below - makes it extremely challenging for large hydrophilic molecules like nucleic acids to efficiently enter the uncompromised skin. We report an amphipathic peptide Mgpe9 (CRRLRHLRHHYRRRWHRFRC) that can penetrate the uncompromised skin, enter skin cells and deliver plasmid DNA efficiently as nanocomplexes in vitro and in vivo without any additional physical or chemical interventions prevalent currently. We observe efficient gene expression up to the highly proliferating basal layer of the skin without observable adverse reactions or toxic effects after delivery of reporter plasmids. The entry mechanism of nanocomplexes possibly involves reversible modulation of junction proteins accompanied by transient changes in skin structure. This peptide holds potential to be used as an efficient transporter of therapeutic nucleic acids to the skin.