Biology subjects

Funk, G.

Publications and source records attributed to Funk, G..

2 recordsLinked to original sources

bioRxiv2024

Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9 with functional confirmation

CRISPR-Cas9 editing is now the leading method for genome editing and is being advanced for the treatment of human disease. CRIPSR editing could have many applications for treatment of neurological diseases, including pain but traditional viral vector delivery approaches have neurotoxicity limiting their use. Overcoming these issues could open the door for genome editing treatments for diseases like intractable pain where the dorsal root ganglia (DRG) would be the desired target. To this end, we describe a simple method for viral-vector-independent transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. As proof of principle, we edited TRPV1, NTSR2, and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successful as demonstrated by the expression of the reporters as early as two days in vitro. CRISPR-Cas9 editing was confirmed at the genome level with insertion and deletion detection system T7-endonuclease-I assay; protein level with immunocytochemistry and Western blot; and functional level through capsaicin-induced Ca2+ accumulation in a high-throughput compatible fluorescent imaging plate reader (FLIPR) system. This work establishes a reliable, target specific, non-viral CRISPR-Cas9-mediated genetic editing in primary human neurons with potential for future clinical application for intractable pain. TeaserWe describe a non-viral transfection method for CRISPR-Cas9 gene editing in human dorsal root ganglion neurons.

neuroscience↗

bioRxiv2023

NaV1.7 mRNA and protein expression in putative projection neurons of the human spinal dorsal horn

NaV1.7, a membrane-bound voltage-gated sodium channel, is preferentially expressed along primary sensory neurons, including their peripheral & central nerve endings, axons, and soma within the dorsal root ganglia and plays an integral role in amplifying membrane depolarization and pain neurotransmission. Loss- and gain-of-function mutations in the gene encoding NaV1.7, SCN9A, are associated with a complete loss of pain sensation or exacerbated pain in humans, respectively. As an enticing pain target supported by human genetic validation, many compounds have been developed to inhibit NaV1.7 but have disappointed in clinical trials. The underlying reasons are still unclear, but recent reports suggest that inhibiting NaV1.7 in central terminals of nociceptor afferents is critical for achieving pain relief by pharmacological inhibition of NaV1.7. We report for the first time that NaV1.7 mRNA is expressed in putative projection neurons (NK1R+) in the human spinal dorsal horn, predominantly in lamina 1 and 2, as well as in deep dorsal horn neurons and motor neurons in the ventral horn. NaV1.7 protein was found in the central axons of sensory neurons terminating in lamina 1-2, but also was detected in the axon initial segment of resident spinal dorsal horn neurons and in axons entering the anterior commissure. Given that projection neurons are critical for conveying nociceptive information from the dorsal horn to the brain, these data support that dorsal horn NaV1.7 expression may play an unappreciated role in pain phenotypes observed in humans with genetic SCN9A mutations, and in achieving analgesic efficacy in clinical trials.

neuroscience↗