Gene Delivery into Primary Neurons and Glia

Primary neurons are useful for understanding the mechanisms underlying many diseases related to the nervous system. Astrocytes are the most numerous glial cells in the brain and are integral in maintaining blood-brain barrier (BBB) integrity, central nervous system (CNS) homeostasis, and synaptic transmission. The potential of gene therapy to restore normal cellular functions has raised the interest for the treatment of devastating diseases such as neurodegenerative diseases. Till now, a wide variety of gene delivery techniques have been developed for in vitro and in vivo studies.

Gene Delivery into Primary Cerebral Cortical Neurons by Lentiviral Vector

The lentiviral vector has been successfully used as an efficient tool for gene transfer in primary cells, including primary human skin fibroblasts, embryonic motoneurons, hippocampal neurons, and cerebral cortical neurons (using EIAV), and neonatal rat ventricular myocytes. In addition to being a useful tool for in vivo gene delivery, lentiviral vectors can also be used for in vitro studies. Cerebral cortical cells cultured in the neurobasal medium are mainly composed of neurons. This purified neuronal cell population combined with lentiviral vectors as a gene delivery tool may make it a good candidate for safe use in in vitro studies of the function of different gene products in cerebral cortical neurons.

Fig.1 Neurons are infected by EGFP-expressing lentiviral vector. (Zhang, 2006)

Arginine-Modified Polymers Facilitate Gene Delivery to Primary Human Astrocytes

Polymeric nanoparticles (NPs) are advantageous for gene delivery due to their low immunogenicity, high biocompatibility, versatility, and protection against peripheral and intracellular degradation of the encapsulated nucleic acids. Poly (lactide-co-glycolide) polymers (PLGA) and polyethylenimine (PEI) are two extensively investigated polymers for drug/gene delivery applications. By combining transfection with arginine-modified PEI-based polymers (AnPn), scientists resolved PLGA-NP-mediated gene delivery to astrocytes while ensuring biocompatibility in both astrocytes and neurons.

Fig.2 Obstacles for gene delivery to CNS. (Proulx, 2020)

Research (solid lines) has identified an improved method that can enhance NP uptake, pDNA nuclear trafficking, and exogenous gene expression in astrocytes. Furthermore, PLGA-A5P10-mediated delivery was cytocompatible with primary human astrocytes and neurons, which supports safe in vivo translation of this NP delivery system.

Dual-Modified Liposome for Targeted and Enhanced Gene Delivery into Mice Brain

The development of neuropharmaceutical gene delivery systems requires strategies to obtain efficient and effective brain targeting as well as BBB permeability. A brain-targeted gene delivery system based on transferrin and cell-penetrating peptide dual functionalized liposome, CPP-Tf-liposome, was designed.

The dual-functionalized liposomes effectively transposed the in vitro barrier model followed by transfecting primary neurons. Liposome tissue distribution in vivo indicated the superior ability of kFGFTf-liposomes to overcome BBB and reach the brain of the mice after single IV administration. Also, the ability of dual-modified liposomes to transfect primary neurons after crossing the in vitro BBB model was assessed. MelTf-lip, kFGFTf-lip, and PasR8Tf-liposomes were able to induce similar expression of GFP in primary neurons.

Fig.3 GFP expression in primary neurons transfected with MelTf-lip, kFGFTf-lip, and PasR8Tf-liposomes. (Rodrigues, 2020)

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References

1. Zhang, Y.; et al. Gene delivery into primary cerebral cortical neurons by lentiviral vector. Cell Biol Int. 2006 Oct;30(10): 777-83.
2. Proulx, J.; et al. Arginine-Modified Polymers Facilitate Poly (Lactide-Co-Glycolide)-Based Nanoparticle Gene Delivery to Primary Human Astrocytes. Int J Nanomedicine. 2020, 15: 3639-3647.
3. Rodrigues, B.; et al. Dual-Modified Liposome for Targeted and Enhanced Gene Delivery into Mice Brain. Journal of Pharmacology and Experimental Therapeutics. 2020, 374(3): jpet.119.264127.